The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Recently, we developed a method for labeling inhibitory synapses in vivo and simultaneously monitored inhibitory synapse and dendritic spine remodeling across the entire dendritic arbor of cortical L2/3 pyramidal neurons in vivo during normal and altered visual experience. In this proposal we seek to characterize with high temporal resolution the nature of the coordinated insertion and removal of excitatory synapses and neighboring inhibitory synapses in the neocortical circuit. To this purpose we will implement our newly developed three-color labeling system combined with spectrally resolved two-photon microscopy to independently and simultaneously monitor postsynaptic markers rand presynaptic afferents representing the full synaptic complement onto individual L2/3 pyramidal neurons in mouse visual cortex. In Aim 1, we monitored the temporal sequence of inhibitory and excitatory synapse remodeling in vivo across the full dendritic arbor of L2/3 pyramidal neurons at short time intervals. For Aim 2, we monitored the effects of experience-dependent plasticity on coordination of inhibitory and excitatory synapse remodeling. Aims 1 & 2 are now essentially complete. This supplement expands on Aim 3, where we examine the specificity of excitatory and inhibitory afferent innervation onto the pyramidal cell dendritic arbor, dependent on location and dynamic behavior. Sex hormones are known to influence the development of inhibition, and estrogen receptors are expressed primarily in the fast-spiking interneurons of the cortex. It is also well established that hormonal treatment alters cortical spine density and turnover in both male and female mice, and that spine density varies in the hippocampus at different stages of the estrus cycle in mature female mice. With this supplement we will extend our ongoing studies on the inhibitory and excitatory afferents targeting L2/3 pyramidal cells to compare distribution and dynamics of specific inputs between males and females. In Aim 4, we are developing and implementing spectrally resolved multiphoton microscopy to enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals. This aim is still in progress, but as an instrument development aim, will not involve animal data collection.